Genetic inhibition of glutamate allosteric potentiation of GABAARs in mice results in hyperexcitability, leading to neurobehavioral abnormalities

Abstract The imbalance between neuronal excitation and inhibition (E/I) in neural circuit has been considered to be at the root of numerous brain disorders. We recently reported a novel feedback crosstalk between the excitatory neurotransmitter glutamate and inhibitory γ‐aminobutyric acid type A receptor (GABAAR)‐glutamate allosteric potentiation of GABAAR functions through a direct binding of glutamate to the GABAAR itself. Here, we investigated the physiological significance and pathological implications of this cross‐talk by generating the β3E182G knock‐in (KI) mice. We found that β3E182G KI, while had little effect on basal GABAAR‐mediated synaptic transmission, significantly reduced glutamate potentiation of GABAAR‐mediated responses. These KI mice displayed lower thresholds for noxious stimuli, higher susceptibility to seizures and enhanced hippocampus‐related learning and memory. Additionally, the KI mice exhibited impaired social interactions and decreased anxiety‐like behaviors. Importantly, hippocampal overexpression of wild‐type β3‐containing GABAARs was sufficient to rescue the deficits of glutamate potentiation of GABAAR‐mediated responses, hippocampus‐related behavioral abnormalities of increased epileptic susceptibility, and impaired social interactions. Our data indicate that the novel crosstalk among excitatory glutamate and inhibitory GABAAR functions as a homeostatic mechanism in fine‐tuning neuronal E/I balance, thereby playing an essential role in ensuring normal brain functioning.

the novel crosstalk among excitatory glutamate and inhibitory GABA A R functions as a homeostatic mechanism in fine-tuning neuronal E/I balance, thereby playing an essential role in ensuring normal brain functioning.

K E Y W O R D S
epilepsy, excitation-inhibition balance, GABA A receptor, synaptic transmission

INTRODUCTION
Neuronal excitation-inhibition (E/I) balance is fundamental for all aspects of brain functions. Disrupting the balance is believed to be at the root of pathogenesis of neurological illnesses including acute brain dysfunctions like stroke and epilepsy, chronic neurodegenerative diseases like Alzheimer's disease, and mental disturbances like schizophrenia and autism. [1][2][3][4][5] In the mammalian central nervous system, synaptic excitation is largely mediated by the principal excitatory transmitter glutamate acting at ionotropic glutamate receptors; whereas synaptic inhibition is largely mediated by inhibitory transmitter γ-aminobutyric acid (GABA) acting on ionotropic GABA A receptor (GABA A R). Understanding the mechanisms that control neuronal balance between glutamate excitation and GABA inhibition is always an extensively researched subject in neuroscience, with scientific and clinical significance. The GABA A R is a ligand-gated heteropentameric chloride ion channel receptor that is assembled from several families of subunits, including α1-6, β1-3, γ1-3, δ, ε, θ, π, and ρ1-3. [6][7][8][9] Most GABA A Rs are assembled from two α subunits, two β subunits, and one γ subunit. 10 In our recent report, 11 using a combination of ligand binding assays, site-directed mutations, and electrophysiological characterizations in human embryonic kidney (HEK) cells overexpressing recombinant GABA A Rs, we identified a novel glutamate binding site in the GABA A R at a pocket located in the interface of α + and β − subunits of the receptor and upon binding, glutamate allosterically potentiates the function of the GABA A R. Given that this glutamatemediated allosteric potentiation of GABA A Rs could be demonstrated in primary neurons, as evidenced by glutamate potentiation of both inhibitory postsynaptic currents (IPSCs) and inhibitory tonic currents, we hypothesized that this novel glutamate-GABA A R crosstalk, that is, the allosteric potentiation of GABA A R by glutamate, may function as an essential homeostatic feedback mechanism of fine-tuning neuronal E/I balance, thereby having major physiological and/or pathological consequences. Indeed, mice with β2 E181G GABA A R subunit knock-in (KI) inhibited glutamate potentiation of GABA A R activity while maintaining baseline GABAAR-mediated synaptic currents, and this mouse line had abnormal phenotypes of sensory process, as well as social interactions; and a pathological condition exhibiting increased kainic acid (KA)-induced seizure activity. 11 The presence of a β subunit is critical for the formation of functional native GABA A Rs. Importantly, the critical pocket-forming glutamic acid residue is conserved among all β subunits (E182 in β2 and β3, and E181 in β1) and single mutation of this residue into alanine almost abolishes glutamate modulation without affecting normal GABA A R function. 7 The β3 subunit is more broadly expressed than the β2 subunit in the mammalian brain, including cerebral cortex, hippocampus and hypothalamus, 12,13 and serves as one of the most important subunits regulating GABA A R function at both circuit 14 and behavioral levels. 1,2,15,16 Therefore, we hypothesized that mice harboring glutamate-binding deficient GABA A Rs generated by KI of β3 Glutamic acid 182 to Glycine (E182G) mutation would have significantly diminished glutamate potentiation on most native GABA A Rs, thereby exhibiting overexcitation phenotypes.
Here, our findings reveal that β3 E182G single point mutation is adequate to largely eliminate the glutamate potentiation of GABA A R responses, and to cause electrophysiological and behavioral abnormalities. More importantly, we also report that bilateral hippocampal wild type β3-containing GABA A Rs overexpression is sufficient to rescue the deficits of glutamate potentiation of GABA A R-regulated responses along with hippocampusrelated behavioral abnormalities. The present study not only provides additional support for a newly identified glutamate allosteric potentiation of GABA A Rs but most importantly demonstrates its importance as a physiological and pathophysiological mechanism by which neuronal E/I balance is controlled in mammalian brains.

β3 E182G mutation impairs glutamate potentiation of GABA A R-mediated responses
To examine the physiological and pathological roles of this glutamate-GABA A R crosstalk in intact animals, we generated KI mice with wild type (WT) endogenous β3 subunits replaced with mutated β3 E182G subunits ( Figure 1A). This mutation changed codon 182 from guanine-adenineguanine (GAG) to guanine-guanine-guanine (GGG), resulting in the amino acid change of glutamic acid to glycine (E182G) of β3. Then deoxyribonucleic acid (DNA) genotyping was used to confirm the successful production of β3 E182G -KI ( Figure 1B). We found that homozygous KI mice were fully fertile, along with a slightly slower growth rate (homozygous KI mice were approximately 10% lighter compared to WT mice) ( Figure S1).
To electrophysiologically characterize the impact of the mutation on GABA A R function, we performed whole-cell patch-clamp recordings of hippocampal CA1 cells from both homozygous KI and WT littermate mice at the age of postnatal day 90. We first determined if the mutation would affect basal GABA A R function through recordings of pharmacologically isolated miniature IPSCs (mIPSCs) and spontaneous inhibitory post-synaptic current (sIPSCs) mediated by GABA A Rs. The results showed that the mutation had no effect on basal mIPSCs and sIPSCs, as reflected by no alterations of their amplitude (for mIPSCs: WT: 24.31 ± 1.17pA, n = 32; KI: 24.62 ± 1.28pA, n = 24; p = 0.860; Figure 1C--E; for sIPSCs: WT: 36.23 ± 3.39pA, n = 17; KI: 37.89 ± 2.67pA, n = 16; p = 0.705; Figure 1G--I) and frequency (for mIPSCs: WT: 2.44 ± 0.21 Hz, n = 32; KI: 2.94 ± 0.28 Hz, n = 24; p = 0.155; Figure 1F; for sIPSCs: WT: 2.92 ± 0.25 Hz, n = 17; KI: 3.19 ± 0.39 Hz, n = 16; p = 0.557; Figure 1G) compared with WT. To directly test the changes in chloride homeostasis of hippocampal CA1 cells, the GABA reversal potential (E GABA ) was measured. The results showed that there was no significant difference in E GABA between WT (−56.49 ± 1.74, n = 15; Figure 1K--M ) and KI (−55.84 ± 1.46, n = 14; p = 0.782; Figure 1K--M ) mice. Thus, consistent with the observations made with the mutation in the recombinant expression system, 11 knocking in the single E182G at the β3 subunit appeared to have no significant impact on the basal function of synaptic GABA A Rs.

β3 E182G KI mice display enhanced learning and memory
The above observations in in-vitro slice preparations confirm a significant reduction in glutamate allosteric potentiation of GABA A R function in KI-β3 E182G mice. Compromising glutamate-GABA A R negative feedback mechanism may lead to behavioral abnormalities in these KI mice. We first examined if there was any alteration in cognitive abilities of KI mice using novel object recognition task and  Barnes maze assessments ( Figure 3). In the novel objective recognition test, we found that both recognition index (RI) number (WT: 0.56 ± 0.04, n = 11; KI: 0.64 ± 0.02, n = 13; p = 0.038; Figure 3A) and time (WT: 0.57 ± 0.04, n = 11; KI: 0.68 ± 0.03, n = 13; p = 0.019; Figure 3B) were significantly increased in KI mice compared to WT controls, suggesting an enhanced object recognition in KI mice. Similar results were also observed in Barnes maze test ( Figure 3C-G). During the acquisition phase, KI mice (n = 16), compared with WT control (n = 12), showed significantly shorter escape latencies (p = 0.044, KI vs. WT; Figure 3C) and smaller error numbers (p = 0.005, KI vs. WT; Figure 3D) to find the escape hole. On memory retrieval test, the KI mice showed significantly better performance than the WT controls as evidenced by the increased correct ratio to find the escape hole (WT: 17.57 ± 2.24%, n = 12; KI: 26.59 ± 3.09%, n = 16; p = 0.036; Figure 3E,F), although there was no group difference in latency to find the escape hole for the first time (WT: 23.69 ± 6.98s, n = 12; KI: 13.01 ± 2.11s, n = 16; p = 0.091; Figure 3G). Taken together, these findings indicate that the performance of cognitive abilities assessed by the two tests is not impaired but enhanced in KI mice.

β3 E182G KI mice exhibit higher neuronal excitability characteristics
By disrupting glutamate-GABA A R crosstalk-mediated negative feedback in controlling neuronal excitability, the β3 E182G KI mice are expected to exhibit certain neuronal overexcitation phenotypes. As predicted, in comparison with WT mice, KI mice showed neuronal hyper-excitability, as evidenced by a reduced threshold for noxious mechanical (WT: 2.79 ± 0.32 g, n = 16; KI: 1.83 ± 0.22 g, n = 23; p = 0.021; Figure 4A) and temperature stimulations (WT: 50.12 ± 5.47s, n = 12; KI: 37.23 ± 3.35s, n = 14; p = 0.049; Figure 4B). Then the well-characterized KA (20 mg/kg; i.p.) mouse model of epilepsy was introduced to further corroborate the higher neuronal network excitability characteristics of the KI mice. These findings revealed that in comparison to WT mice KA-induced seizure activity was significantly increased in KI mice, as evidenced by reduced latency (WT: 189.64 ± 15.25s, n = 11; KI: 58.33 ± 4.67s, n = 12; p < 0.001; Figure 4C) and increased severity (p < 0.001; Figure 4D) of KA-induced seizure activity. These results not only further support F I G U R E 3 β3 E182G knock-in (KI) mice exhibit enhanced learning and memory in both novel object recognition and Barnes maze tests. (A and B) In novel object recognition test, both number (A) and time (B) of the recognition index (RI) are increased in KI mice (KI; n = 13), compared with WT mice (WT; n = 11). (C-G) In the Barnes maze test, both latency (C) and error number (D) to find the escape hole are significantly decreased in KI mice (KI; n = 16), compared with WT mice (WT; n = 15) during learning phase. During the probe phase, the correct ratio (E and F) is significantly increased in KI mice, compared with WT mice, and the latency to the first entry to the escape hole (G) shows a trend of decrease. Data are expressed as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001. that the KI mice have a deficit in glutamate potentiation of GABA A R function but also provide additional evidence for a critical negative feedback role of this modulation in controlling neuronal E/I balance.

β3 E182G KI mice display autistic-like behaviors
Previous studies have revealed that the disruption of E/I balance contributes to the pathogenesis of autism spectrum disorders (ASD). [16][17][18][19][20] We, therefore, investigated if an alteration of autistic-like behaviors occurred in the β3 E182G KI mice by evaluating their performances in social interaction and locomotor activity. In a three-chambered social interaction test with a mouse (M) versus a toy (T), we found that the KI mice displayed obvious social deficits as reflected by a significant reduction in both number (WT: 1.24 ± 0.12, n = 16; KI: 0.92 ± 0.07, n = 13; p = 0.026; Figure 5A,B) and time (WT: 1.33 ± 0.24, n = 16; KI: 0.67 ± 0.12, n = 13; p = 0.024; Figure 5A,C) of the social index (SI), compared to WT controls. Immediately after test, the test mouse was co-housed with the paired mouse during test for 24 h to familiarize with it (FM). Twenty-four hours later, the second test was performed to determine the social novelty preference in the KI mice.
During this test, we replaced the toy with another ageand gender-matched stranger mouse (SM). These results again showed that compared with WT controls, the KI mice exhibited a markedly reduced SI in both number (WT: 1.02 ± 0.13, n = 14; KI: 0.64 ± 0.08, n = 15; p = 0.026; Figure 5D,E) and time (WT: 2.02 ± 0.38, n = 14; KI: 0.75 ± 0.12, n = 15; p = 0.008; Figure 5D,F). We next evaluated potential changes in locomotor activity using an open-field test (Figure 5G-J). As we expected, spontaneous locomotor activity in KI mice was significantly increased, as evidenced by the increased total traveling distance during the test (WT: 12.83 ± 2.26 m, n = 11; KI: 22.40 ± 2.19 m, n = 15; p = 0.006; Figure 5G,H). Surprisingly, this increased locomotor activity appeared to be associated with a reduced level of anxiety. The results showed that the KI mice displayed increased numbers of entry into the center zone (WT: 11.00 ± 3.05, n = 11; KI: 24.40 ± 3.27, n = 15; p = 0.008; Figure 5G,I) and more time (WT: 14.77 ± 4.75s, n = 11; KI: 48.47 ± 7.39s, n = 15; p = 0.002; Figure 5G,J) in the center zone of the testing chamber compared with their WT counterparts. Such reduced anxiety phenotype was not expected as neuronal overexcitation due to disrupted E/I imbalance is often associated with an increased anxiety. 21 We, therefore, further assessed the alteration of anxiety level in the KI mice using the elevated plus maze test ( Figure 5K

Hippocampal expression of α1β3 reverses the impairments of glutamate allosteric potentiation of GABA A Rs in β3 E182G KI mice
To determine if the impairment of glutamate allosteric modulation of GABA A Rs and behavioral changes in the KI mice are results of abnormal development or dynamic alteration of neuronal E/I balance due to the point mutation of residue E182 of β3, we overexpressed functional WT β3 subunit-containing GABA A Rs in hippocampal neurons by bilateral intra-hippocampal microinjection of adenoassociated viruses carrying WT β3 (AAV β3 ), and examined its ability to rescue the electrophysiological and behavioral phenotypes in the KI mice. Since topically expressed single GABA A R subunit may not efficiently heteromerize with the endogenous GABA A R subunits, 22 we also cotransfected neurons with the α1 (AAV α1 ) to facilitate the formation of α1 and β3 recombinant receptors (AAV α1β3 ), thereby complementing β3 E182G -containing GABA A Rs in the KI mice. We first studied the functional rescue of glutamate allosteric potentiation of GABA A Rs in hippocampal slices. We recorded the hippocampal CA1 pyramidal neurons that were co-expressing fluorescently identifiable recombinant GABA A Rs containing both α1 (Green) and β3 (Red) ( Figure S2). These results showed that overexpressing α1β3 recombinant GABA A Rs fully restored the glutamate-mediated allosteric potentiation of GABA A R function ( Figure 6A,D), as evidenced by the significant potentiation of GABA A R-mediated wholecell currents by bath application of L-Glu (KI+AAV α1β3 ; 50 μM; n = 13; Figure 6A) or D-AP5 (KI+AAV α1β3 ; 50 μM; n = 22; Figure 6B), and GABA A R-mediated IPSCs by TBS (KI+AAV α1β3 ; T1-T5; n = 13; Figure 6C,D). The functional rescue appeared to be due to the expression of WT β3-containing GABA A Rs that enabled the glutamate-GABA A R feedback cross-talk, and not a result of simply increased numbers of GABA A R, since there was no significant difference in amplitude, frequency of mIPSPs between neurons infected with (KI+AAV α1β3 ; Figure 6E,H) and without (KI; Figure 6E,H) AAV α1β3 recombinant GABA A Rs. These results indicate that overexpressing WT β3 subunit-containing GABA A Rs in the KI mice is sufficient to rescue the deficient glutamate allosteric potentiation of GABA A R-mediated neuronal inhibition without affecting the receptor-mediated basal synaptic transmission.

2.6
Hippocampal expression of α1β3 subunits of GABA A Rs rescues behavioral abnormalities in β3 E182G KI mice Based on the ability of overexpressing α1β3 subunits of GABA A Rs to restore the defective glutamate-GABA A R feedback cross-talk in the KI mice, we predicted that the bilateral hippocampal infection of AAV α1β3 should also be able to rescue some of the hippocampus-related abnormal phenotypes in the β3 E182G KI mice, particularly the increased KA-induced seizure activity and impaired social interactions. Consistent with our reasoning, we found that overexpression of α1β3 (KI+AVV α1β3 , n = 14; Figure 7A,B) significantly reduced epileptic seizures, compared to KI mice (KI, n = 8; Figure 7A,B). There was a significant increase in the latency (KI: 47.88 ± 2.35; n = 8; KI+AAV α1β3 : 131.14 ± 19.62s; n = 14; p = 0.001; Figure 7B) and decrease in the severity (KI+AAV α1β3 : n = 14; KI: n = 8; p = 0.001; Figure 7B) of KA-induced seizure activity in the KI mice infected with AVV α1β3 . Similarly, we found  Taken together, these findings demonstrate that expression of recombinant GABA A Rs containing the WT β3 subunits can rescue electrophysiological and behavioral phenotypes due to β3 E182G mutation-induced disruption of glutamate-GABA A R negative feedback cross-talk. In addition, the results strongly suggest that some of the hippocampusrelated behavioral abnormalities in these KI mice are the results of functional, rather than developmental, alteration of the glutamate-GABA A R feedback loop as they can be rescued at adulthood.

DISCUSSION
Maintaining a proper neuronal E/I balance is fundamental for brain functioning. Despite intensive studies over the last few decades, detailed mechanisms regulating E/I balance in mammalian brains remain poorly understood. In our recent study, 11 we have identified a novel glutamatebinding site on the GABA A R, at which glutamate acts to allosterically potentiate GABA A R function. The presence of such a functional cross-talk between the principle excitatory transmitter glutamate and the major inhibitory GABA A R predicts a critical homeostatic feedback role of the crosstalk in fine-tuning neuronal E/I balance. With no specific antagonist to inhibit glutamate from binding to this newly identified site on GABA A R, we have examined the potential involvement of this cross-talk under both physiological and pathological conditions through the generation of a β3 E182G KI mouse line in which the glutamate binding pocket in most of the native GABA A Rs is largely inhibited. β3 E182G mutation is artificial and has not been found in the human brain. We provide direct evidence that genetically impairing this glutamate-GABA A R cross-talk disrupts neuronal E/I balance, leading to neurophysiological and behavioral phenotypes with characteristics of hyper neuronal excitability. Thus, our work demonstrates that through a homeostatic feedback mechanism, this newly identified glutamate-GABA A R crosstalk plays an indispensable role in fine-tuning neuronal E/I balance under both physiological and pathological conditions. The co-release of glutamate with GABA at GABAergic terminals and subsequent glutamate allosteric potentiation of adjacent GABA receptors plays important roles in some physiological and pathological processes. 23,24 Under certain conditions, such as increased synaptic activities during the production of certain forms of synaptic plasticity or following ischemic brain insults, extracellular glutamate concentrations can reach levels close to or even above the EC50, which may lead to glutamate allosteric potentiation of the adjacent GABA A Rs. 25 This can result in increased GABA A R-mediated neuronal inhibition and counteract glutamate receptor-mediated overexcitation. The relatively high EC50 for the glutamate-mediated allosteric potentiation of GABA A Rs is an important feature that ensures glutamate functions as an excitatory transmitter mediating synaptic transmissions at the vast majority of excitatory synapses under most physiological conditions. However, under conditions of overexcitation of glutamatergic neurons and/or compromised glutamate uptake mechanisms, the cross-talk between glutamate and GABA can potentially bear physiological and pathological significance. Therefore, understanding the mechanisms underlying the co-release of glutamate and GABA and their functional implications is crucial for the development of effective treatments for various neurological disorders.
The GABA A R is a ligand-gated heteropentameric chloride ion channel that is assembled from families of subunits, including α1-6, β1-3, γ1-3, δ, ε, θ, π, and ρ1-3. [6][7][8] The majority of the native GABA A Rs are formed by the assembly of two α subunits, two β subunits and one γ subunit with two GABA binding sites. 10 In particular, β3 subunit-containing GABA A R is broadly expressed in the brain, including cerebral cortex, hippocampus, and hypothalamus. [26][27][28] Therefore, the β3-containing GABA A Rs have been shown to have important roles in several pathophysiologic processes, such as epilepsy, 29,30 Angelman syndrome and Prader-Willi syndrome. 31,32 Consistent with the involvement of the critical glutamic residue E182 in β3 (E182 and E181 for β1 and β2, respectively), we identified in recombinant GABA A Rs, 11 the β3 E182G KI mice, had no effect on fertility but grew slightly slower, exhibit impaired glutamate-GABA A R crosstalk, as evidenced by the significantly reduced potentiation of GABA A R-mediated responses by exogenously applied glutamate and glutamate-like ligand AP5, in comparison with that in the wildtype counterparts. These results not only provide further evidence supporting the functional operation of the newly identified glutamate-GABA A R cross-talk in wild-type animals but also confirm the successful disruption of the cross-talk in the KI mice.
Neurophysiological and pathological phenotypes observed in these β3-specific glutamate-GABA A R crosstalk deficient mice reveal the important physiological and pathological roles of this cross-talk in vivo. TBS induces much greater GABA A R-mediated IPSCs in WT mice than in β3 E182G KI mice, which suggests the stimulation causes neurons to fire at high frequency and results in an increased concentration of glutamate endogenously being released from their axonal terminals. The fact that the potentiation is absent in the glutamate-GABA A R cross-talk compromised β3 E182G KI mice strongly suggests that the theta-burst stimulation-induced enhancement of GABA A R function is indeed mediated by the glutamate-GABA A R cross-talk. Since theta-burst is one of the common neuronal rhythms that occur in the brain under physiological conditions, our results strongly argue for the functional engagement of the cross-talk as a homeostatic feedback mechanism under physiological conditions. Knocking in the single E182G at the β3 subunit appeared to have no significant impact on the basal function of synaptic GABAARs, including sIPSC and mIPSC. Notably, the tonic inhibitions, which are essential for maintaining neuronal excitability, are mainly mediated by these extrasynaptic. Further identification of the tonic inhibition would be of great significance for GABA A Rs' basal function in the future.
Compromising the function of this homeostatic feedback is expected to lead to neuronal overexcitation phenotypes. This is supported by two lines of evidence presented in the current study. First, in comparison with the wild type counterparts, the KI mice showed a significant decrease in their thresholds to nociceptive stimulations to the limbs ( Figure 4A,B). While GABA A Rs in the central nucleus of the amygdala have an important role in pain control, 33,34 the findings strongly support that glutamate allosteric potentiation of GABA A R is an essential process in GABA A R mediated pain controls. Second, intraperitoneal injection of KA at the same concentration induced more severe seizures with increased severity and shortened latency in the KI mice than in the wildtype controls ( Figure 4C,D). Meanwhile β3 E182G KI mice had more severe seizures with elevated severity and reduced latency than the β2 E181G KI mice, indicating that the single E182G mutation at the β3 subunit appears to increase neuronal network excitability phenotype compared to E181G mutation in the β2 subunit. These results not only provide further evidence for the widely accepted roles of GABA A Rs in epileptogenesis 14,35 but also land additional support for the physiological and pathological significance of the glutamate-GABA A R cross-talk in fine-tuning E/I balance, and thereby controlling neuronal excitability.
Many previous studies have reported that the E/I balance plays critical roles in normal brain functions; accumulating studies are showing that disruption of this balance may cause neuropsychiatric disorders, such as autism. [16][17][18][19] Consistent with these findings, we observed in the present study that the β3 E182G KI mice exhibit increased locomotor activity and impaired social interaction ( Figure 5A-F), which are the two major phenotypes associated with autistic spectrum disorders. However, it is interesting to note that the KI mice unexpectedly exhibit reduced anxiety-like behavioral phenotype ( Figure 5G-L) and enhanced hippocampus-related spatial learning and memory (Figure 3). One possibility is that due to the disrupted glutamate-GABA A R cross-talk, β3 E182G mutation impairs the ability of the GABA A R to adaptively increase its function in response to enhanced glutamate transmission. The inability to adaptively increase GABAergic inhibition results in a relative deficit in GABA A R-mediated inhibition, contributing to the reduced anxiety and increased learning ability. Indeed, previous studies have shown that the GABA A receptor antagonist bicuculline is able to improve both spatial learning and working memory, and decrease anxiety in hyperammonemic rats. 13 Nonetheless, our study provides strong evidence that disruption of this homeostatic feedback mechanism mediated by the glutamate-GABA A R cross-talk leads to atypical phenotypes of autistic spectrum disorders.
The majority of phenotypes in β3 E182 mice are hippocampus-dependent, we therefore overexpressed recombinant GABA A Rs containing the WT β3 subunits in hippocampal neurons and examined its ability to rescue the electrophysiological and behavioral phenotypes in the KI mice. As expected, expression of functional WT β3 subunit-containing GABA A Rs can rescue electrophysiological and behavioral phenotypes, further indicating that the impairment of glutamate allosteric potentiation of GABA A Rs and behavioral changes in the KI mice are due to the point mutation of residue E182 of β3.
Notwithstanding these contributions, however, there are some limitations. Social and communication deficits are typical symptoms of ASD, 36 and many studies are showing that suppressed GABAergic inhibition is a common feature of the autistic brain. 37 Additionally, ASD is often accompanied by other mental diseases, such as hyperactivity and anxiety. 38 The β3 E182G KI mice exhibited hyperactivity in the open field test and elevated plus maze test but did not display anxiety-related phenotype. Although, the phenotype of β3 E182G KI animal model differs somewhat different from the human condition, additional behavioral assessments would have to be conducted to verify the anxiety-related phenotypes of β3 E182G KI in the future. Furthermore, given the high comorbidity of seizure disorders and autism, 37 although the severity of KA-induced seizure activity was significantly increased in KI mice, further electroencephalogram (EEG) recordings of β3 E182G KI mice with or without KA-induced seizure to explore more underlying mechanisms in the future.
Taken together, our results provide strong evidence supporting the mechanistic and functional framework of our newly identified glutamate-GABA A R cross-talk. 11 This cross-talk plays a homeostatic role in maintaining a proper balance between glutamate-mediated neuronal excitation and GABA-mediated neuronal inhibition under both physiological and pathological conditions. Disruption of this cross-talk will lead to various phenotypes associated with neuronal hyperexcitability. Characterization of this crosstalk in detail can be expected to shed more light into mechanisms of how the brain maintains a proper neuronal E/I balance, and how its deficiency contributes to the pathogenesis of certain brain disorders. It may also provide scientific basis upon which new therapeutics can be developed for treating these brain disorders. This previously unrecognized cross-talk between the two principle transmitter systems in the mammalian brain not only blurs the traditional distinction between excitatory and inhibitory transmitters but also necessitates further investigation into its physiological and/or pathological roles.

Animals
Wild-type (WT) and β3 E182G KI mice were kept in a colony room with a temperature-controlled (21 • C) 12-h light/12-h dark cycle, food and water were available ad libitum, and all electrophysiological and behavioral assessments were performed during the light cycle at the age of postnatal day 90. All procedures were performed in accordance with the Chongqing Science and Technology Commission guidelines for animal research and approved by the Children's Hospital of Chongqing Medical University Animal Care Committee.

Adeno-associated virus and microinjection
To express recombinant α1/β3 GABA A Rs in vivo, adenoassociated virus expressing α1 (AAV α1 ) and β3 (AAV β3 ) were constructed by OBiO Technology (Shanghai, China). Titers were 5 × 10 12 TU/ml. After anesthetization with sodium pentobarbital, mice at the age of 2 months old were mounted on a stereotaxic instrument, and 0.8 μl of AAV α1 and AAV β3 were co-microinjected into the dorsal hippocampal CA1 area through a drilled hole (−2.3 mm posterior, ± 2.0 mm lateral and 2.5 mm ventral relative to bregma). One month after AAVs microinjection, the electrophysiological and behavioral tests were performed. For electrophysiological experiment, only the cells that expressed both the α1 and β3 subunits of GARA A R were selected for recordings.

Electrophysiology studies
Mice were deeply anesthetized with urethane ( To induce eIPSCs, a concentric stimulation electrode was placed at the Schaffer collateral pathway and the stimulation intensity was set to 50% of the maximal eIPSC response. CNQX (20 μM) was added to the bath to block AMPA receptors. After obtaining a stable baseline, D-AP5 (50 μM) or L-Glu (50 μM) was added to the recording solution to measure the potentiation of GABA A R-mediated inhibitory currents. Synaptic TBS was delivered, consisting of 5 trains of 4 pulses at 100 Hz, with an inter-train interval of 200 ms. For recordings of mIPSCs (TTX (0.5 μM) was used to block voltage-gated Na + channels) and sIPSCs, CNQX (20 μM) and D-AP5 (50 μM) were added to the recording solution to block action potential and glutamatergic transmission, respectively. GABA reversal potential (E GABA ) recordings were performed using a perforated patch-clamp technique. Recording pipettes (5-6 MΩ) filled with the intracellular solution that contained (mM): K-Gluconate 136.5, KCl 17.5, NaCl 9, EGTA 0.2, HEPES 10, MgCl2 1, pH 7.2; osmolarity, 285 mOsm. Gramicidin at a concentration of 50 μg/ml was used as the pore-forming agent for perforated recordings. Around 20-40 min after giga seal formation, the access resistance slowly dropped and stabilized at ∼40 MΩ. Holding potentials were stepped from −110 to −10 mV. At each holding potential step, GABA (100 μM, 50 ms) was perfused directly onto CA1 pyramidal neurons through a pipette (2-3 mm tip) using a VC3 perfusion delivery system (Plexon). During recording, CNQX (10 μM), D-AP5 (50 μM) and TTX (0.5 μM) were added in extracellular solution to block AMPA receptors and NMDA receptors and voltagegated sodium channels, respectively. All experiments were performed at room temperature.

Pain threshold tests
The mechanical withdrawal thresholds were ascertained to assess mechanical hyperalgesia, as described previously. 33 Each animal was put into an 8 × 9 × 8 cm clear plastic cage with wire mesh to allow the filament to be inserted from below. Prior to testing, the filament was applied to the left hind paw's plantar surface and allowed to acclimatize for at least 10 min. Three measurements were carried out, each at 30-s intervals. The mechanical withdrawal threshold was based on the average value. A hot plate test (Stoelting, Wood Dale, IL, USA) was used to measure thermal hyperalgesia. Mice were individually put on a hot plate heated to 55 ± 0.5 • C. Withdrawal of the paw from the thermal stimulus was measured by the latency from the onset to the time of hind paw jumping or licking. The test has a 90-s cutoff time to prevent harm and damage. Double-blind methods were used to evaluate both pain-related behavioral tests.

KA-induced seizures
A 20 mg/ml concentration of KA was dissolved in sterile saline. Intraperitoneal injections (i.p.) of KA (20 mg/kg) or saline in the same volume as the vehicle control were used to cause seizures. 40 Thirty minutes prior to the injection of KA, diazepam (20 mg/kg, s.c., procured from Children's Hospital of Chongqing Medical University) was administered. A trained observer who was unaware of the mice's genotype or therapy evaluated seizure activity every 15 min for 2 h using the scale as described previously. 40

Social interaction test
Social interaction was evaluated by using a three-chamber apparatus as described previously. 41

Elevated plus maze
The

Novel object recognition test
Mice were placed in the 40 × 40 cm open box for 5 min for adaption 24 h before the test. Mice were placed in the box for 5 min to explore two identical objects on the test day, after which they were returned to their food cages. Two hours later, the mice were returned to the box, where one of the objects was replaced by a novel object. The animals were allowed to explore both objects for another 5 min. The experimenter recorded the occurrence of head dips to the objects in a double-blinded fashion. The recognition index (RI) was calculated by using the equation: RI = number or time spent on novel object/total number or time spent on both objects.

Barnes maze test
The Barnes maze was used to study hippocampus-based spatial learning and memory. The apparatus is made of a 1.2-m diameter white circular platform with 18 holes (5-cm diameter) around the edge, one of which has an escape box underneath. To record the latency and number of errors made while trying to find the escape box, a charge coupled device (CCD) camera was positioned above the maze center. Video outputs were then digitalized using an ANY-Maze Video Tracking System (Stoelting, USA). Animals were given 3 min to acclimatize to the maze 24 h before spatial training. Then, for the following 6 days, the animals underwent two trials per day of training in a spatial learning task with a 15-min interval break. If the mouse was unable to find the box, or if it did find the box but failed to enter it within 5 min, it would be gently guided to the escape box and remained there for 60 s before being returned to its home cage. Twenty-four hours following the last training trial, mice were subjected to a 5-min probing test while the escape box was blocked. The correct ratio was calculated by using the equation as described previously 42 : % correct ratio = number of finding the escape box/total number of reaching all holes × 100%.

Data analysis
Values were expressed as mean ± SEM (n = number of experiments). The data of the Barnes maze training and seizure class were analyzed by a two-way between/withinsubjects factorial analysis of variance (ANOVA), with genotype as the between-subjects factor and training session (time) as the within-subjects factor. All significant main effects and interactions were further analyzed using Turkey's comparisons. Other data were analyzed by a two-tailed student's t-test. All statistical analyses were performed using SPSS 22.0, and GraphPad Prism8.0.2. Statistical significance was set at p < 0.05.

A U T H O R C O N T R I B U T I O N S
YD, YTW, and ZD conceived the study and wrote the manuscript. YD, MW, and QT performed phenotypical analysis and behavioral studies. JL, YP, YW, and DCW performed electrophysiological studies. All authors have read and approved the article.

A C K N O W L E D G M E N T S
The authors are grateful to other members in the Dong laboratory for the technical support and helpful suggestion.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflicts of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

E T H I C S S TAT E M E N T
Animal ethics approval is approved by the Animal Ethics Committee of Children's Hospital of Chongqing Medical University (approval number: CHCMU-IACUC20210114017).